Dig Dis 2005;23:264274 DOI: 10.

1159/000090174

Molecular Mechanisms of Alcohol-Induced Hepatic Fibrosis

Sren V. Siegmund a, c Steven Dooley b, c David A. Brenner a
a b

Department of Medicine, Columbia University College of Physicians and Surgeons, New York, N.Y., USA; Center of Molecular Alcohol Research in Gastroenterology, and c Department of Medicine II (Gastroenterology, Hepatology, Infectious Diseases), University Hospital Mannheim, University of Heidelberg, Mannheim, Germany

Key Words Ethanol Liver brosis Fibrogenesis Hepatic stellate cells Oxidative stress Signal transduction

Introduction

Abstract Alcohol abuse is a major cause of liver brosis and cirrhosis in developed countries. Before alcoholic liver brosis becomes evident, the liver undergoes several stages of alcoholic liver disease including steatosis and steatohepatitis. Although the main mechanisms of brogenesis are independent of the etiology of liver injury, alcoholic liver brosis is distinctively characterized by a pronounced inammatory response due to elevated gutderived endotoxin plasma levels, an augmented generation of oxidative stress with pericentral hepatic hypoxia and the formation of cell-toxic and probrogenic ethanol metabolites (e.g. acetaldehyde or lipid oxidation products). These factors, based on a complex network of cytokine actions, together result in increased hepatocellular damage and activation of hepatic stellate cells, the key cell type of liver brogenesis. Although to date removal of the causative agent, i.e. alcohol, still represents the most effective intervention to prevent the manifestation of alcoholic liver disease, sophisticated molecular approaches are underway, aiming to specically blunt probrogenic signaling pathways in liver cells or specically induce cell death in activated hepatic stellate cells to decrease the scarring of the liver.
Copyright 2005 S. Karger AG, Basel

Liver brosis represents a wound-healing process in response to a variety of chronic injurious stimuli. The most important etiology in Western industrial countries is chronic alcohol abuse [1], accounting for more than 50% of all cirrhotic livers in Western countries [2]. Typically, injury is present for years before signicant scar accumulates. Before alcoholic liver brosis becomes evident, the liver undergoes several stages of alcoholic liver disease (ALD) including steatosis and steatohepatitis. Based on the high regenerating potential of the liver, these stages including early liver brosis can be resolved, and there is some clinical and experimental evidence that even advanced brosis/cirrhosis can be reversible, if the cause of liver injury is eliminated [36]. In this review, we focus on the molecular mechanisms contributing to alcohol-induced liver brosis and cirrhosis as well as on novel molecular treatment strategies.

Molecular Pathogenesis of Alcoholic Liver Fibrosis

Hepatic brosis represents the consequence of a wound-healing response to repeated injury [7, 8]. Fibrosis is characterized by an excessive deposition of extracellular matrix (ECM), and loss of parenchymal tissue. The distribution of brotic tissue depends on the origin of liver injury. In alcohol-induced liver disease, it is located

2005 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com

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David A. Brenner, MD Department of Medicine, Columbia University 630 West 168th Street, PH 8 East Room 105 New York, NY 10032 (USA) Tel. +1 212 305 5838, Fax +1 212 305 9822, E-Mail dab2106@columbia.edu

Normal liver
Macrovesicular steatosis Chronic ETHANOL Low density ECM

Fibrotic liver
Hepatocyte regeneration Hepatocyte apoptosis/ necrosis Interstitial ECM/ fibrillar collagen Basal membrane Fenestrated endothelium Microvilli loss Activated HSCs Defenestrated endothelium

Quiescent HSC Quiescent Kupffer cell

Sinusoid Sinusoid
Activated Kupffer cell/ macrophage

Fig. 1. Schematic representation of alterations in the liver during ethanol-induced brogenesis. Chronic alcoholic injury of the liver causes accumulation of macrovesicular fat in hepatocytes. Quiescent, vitamin A-storing hepatic stellate cells (HSCs) become activated, e.g. triggered by paracrine stimulation from activated Kupffer cells/ macrophages. They transdifferentiate into myobroblast-like cells, producing excessive amounts of extracellular matrix (ECM), mainly brillar collagen in the space of Diss. This leads to defenestration of the sinusoidal epithelium and formation of basal membranes. Furthermore, reactive oxygen species-triggered hepatocyte cell death contributes to HSC activation. HSCs proliferate and migrate towards the site of hepatocellular injury. Finally, ethanol-induced liver cirrhosis features mostly micronodular regeneration of hepatocytes as part of an irregular remodeling of the liver.

in pericentral and perisinusoidal areas, whereas brosis induced by chronic viral hepatitis predominantly occurs around portal tracts [8, 9].

Hepatic Stellate Cell Activation in ALD

Hepatic stellate cells (HSCs; also known as Ito cells, fatstoring cells or hepatic lipocytes) are mainly responsible for increased synthesis and deposition of ECM in the liver during brogenesis [711]. HSCs are perisinusoidal cells that reside in healthy liver in a quiescent state. Their main function is storage and homeostasis of vitamin A and other retinoids, which are retained in cytoplasmatic lipid droplets as retinyl esters together with triacylglycerides. Further functions comprise ECM homeostasis, including production and degradation of normal hepatic ECM, production of apolipoproteins, prostaglandins and cytokines as well as regulation of sinusoidal blood ow [12, 13]. Following a brogenic stimulus, HSCs undergo a complex activation process in which the cells change from a quiescent to an activated myobroblast-like phenotype, and proliferate, migrate to the site of liver injury and produce excessive amounts of ECM resulting in scarring of

the liver. Alcohol-induced activation of HSCs occurs as a response to chronic alcohol uptake with considerable loss of functional hepatocellular tissue, whereas a single acute binge of ethanol does not cause HSC activation [10]. Activation of HSCs (g. 2) can be subdivided into two phases, that is (i) initiation and (ii) perpetuation [10, 11, 13, 14]. Initiation results from paracrine stimulation by impaired neighboring cells, e.g., injured hepatocytes, endothelial cells and activated Kupffer cells, as well as from early and subtle changes in ECM composition. Some authors divide the initiation phase into a pre-inammatory phase before and an inammatory phase after involvement of immune cells [2, 15]. Initiation is associated with transcriptional changes in HSCs involving rapid induction of immediate early genes encoding, e.g., for cytokines or membrane receptors, rendering the cells responsive to cytokines and other local stimuli, such as acetaldehyde. As a consequence, a phenotypical transdifferentiation is evident. The mechanisms of HSC activation originate already in the early stages of ALD, steatosis and steatohepatitis (g. 1). One mechanism by which hepatic steatosis causes HSC activation is generation of ROS produced in meta-

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CHRONIC ALCOHOLIC LIVER INJURY

(ROS, actetaldehyde, aldehyde-protein adducts, lipid peroxides, apoptotic/necrotic hepatocytes, etc.)

Proliferation Contractility

Fibrogenesis PDGF ET-1 TGF- 1 Integrins

Activation

Apoptosis

(Regression?) ROS, various cytokines Endotoxin Activation Chemotaxis (M-CSF, MCP-1) Autocrine selfstimulation of quiescent HSCs MMPs

Matrix degradation

Inflammatory cells (Kupffer cells, neutrophils, T-cells)

Fig. 2. Schematic overview of the different aspects of hepatic stellate cell (HSC) activation and the main cyto-

kines/mediators involved. After activation, the major phenotypic changes include brogenesis, proliferation, contractility, matrix degradation and chemotaxis. Activated HSCs may also undergo apoptosis, representing a major eld of research for anti-brotic therapy. A special aspect represents inammatory cells in alcoholic liver disease (ALD). Reactive oxygen species (ROS) and various cytokines derived from endotoxin-activated inammatory cells are main contributors to HSC activation in ALD. ET-1 = Endothelin-1; MCP-1 = monocyte chemotactic protein; M-CSF = macrophage-colony stimulating factor; MMPs = metallo-matrix proteinases; TGF-1 = transforming growth factor-1.

bolically impaired hepatocytes. Reactive oxygen species (ROS) can cause HSC activation alone or in combination with other factors that may activate HSCs in ALD, such as cytokines or lipid oxidation products [1620]. Persisting alcohol consumption causes a gradual involvement of inammatory cells. Chronic inammatory conditions with an abundance of proinammatory cytokines, including TNF-, IL-1 or IL-6, induce and perpetuate the activation of HSCs [21, 22]. Activated HSCs, in turn, may amplify inammation through release of neutrophil and monocyte chemoattractants and upregulation of adhesion molecules that are required for leukocyte adhesion and transmigration [2123].

Furthermore, the increased serum levels of gut-derived endotoxin most likely contribute directly to the perpetuation of HSC activation in ALD. It has been shown that activated HSCs express the components of endotoxin-recognizing receptors, including CD14, TLR4, and MD2. Endotoxin induces activation of the proinammatory NFB and JNK pathways, followed by expression of chemokines and adhesion molecules in activated human HSCs [24]. ROS, either from hepatocytes or activated Kupffer cells, have an articulate paracrine effect on quiescent HSCs [16, 17] (g. 2). Their activity is further augmented in vivo by depletion of cellular antioxidants like glutathi-

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one, a typical feature of ALD [25]. The probrogenic effect of ROS was conrmed by the nding that ROS-scavenging enzymes, such as the superoxide dismutase system (SOD1, SOD2 and SOD3), prevent ALD in animal models [26, 27]. In vitro experiments emphasize the importance of ROS in the mechanism of HSC activation: medium derived from freshly isolated hepatocytes exposed to oxidative stress increases proliferation and collagen synthesis in cultured HSCs [28]. Moreover, augmented ethanol-induced ROS production in CYP2E1-overexpressing HSCs leads to enhanced collagen 1(I) gene expression [29]. By changing the cellular redox state, ROS also modulate the activity of transcription factors involved in HSC activation and brogenesis, such as c-Jun/ AP-1, NFB, SP1 or c-Myb [30]. Ethanol metabolism modies the cellular redox state in the liver by altering the ratio of NAD/NADH and NADP/NADPH, which leads to increased production and accumulation of lactic acid, another metabolite known to induce HSC activation and brogenesis [31]. Beside CYP2E1 [29, 3134], NADPH oxidase plays an additional key role in oxidant radical formation and activation of HSC. NADPH oxidase, mainly expressed in activated Kupffer cells [16, 17], may activate HSCs by generating H2O2, which functions directly as a second messenger for collagen 1(I) gene upregulation [35, 36]. Recent studies underline the importance of NADPH oxidase-derived ROS in hepatic brogenesis mediated by angiotensin II, a powerful probrogenic cytokine and mitogen [37, 38]. Thus, activated HSCs themselves are a signicant source of ROS [38, 39]. Hepatic hypoxia induced by ethanol metabolism holds another key function in ethanol-related activation of HSCs. Ethanol-induced hypoxia and hepatocellular damage are augmented in pericentral areas of the liver acinus. Hypoxia causes upregulation of the transcription factor hypoxia-inducible factor-1, which upregulates the transcription of the cytokine vascular endothelial growth factor (VEGF), another strong paracrine and autocrine inducer of HSC activation [40, 41]. Ethanol itself, and even more its rst metabolization product, acetaldehyde, may have activating effects on HSCs [31] (g. 2). Acetaldehyde can directly upregulate collagen genes [30, 31, 42]. However, acetaldehyde-derived lipid peroxides, such as 4-hydroxy-nonenal or malondialdehyde, seem rather to promote hepatic brogenesis in already activated HSCs than to initiate HSC activation [19, 4346]. Further, fatty acid ethyl esters (ethanol metabolites generated in a non-oxidative fashion and esteried with fatty acids) activate quiescent HSCs via a

mitogen-activated protein kinase (MAPK)-dependent pathway [47]. Finally, sinusoidal endothelial cells also participate in ethanol-related HSC activation. Injury of sinusoidal epithelial cells by ROS or acetaldehyde stimulates production of certain bronectin isoforms in these cells which then activate HSCs. Moreover, damaged epithelial cells convert latent transforming growth factor (TGF)-, the most brogenic cytokine to date, into its active form through plasmin activation [11].

Mechanisms of Excessive Accumulation and Irregular Deposition of ECM in Ethanol-Induced Liver Fibrosis

Upregulation of collagen synthesis is one of the most striking molecular responses of activated HSCs and is mediated by both transcriptional and post-transcriptional mechanisms. The type I collagen molecule is a heterotrimer composed of two 1(I) chains and one 2(I) chain [48]. The mechanism of collagen 1(I) and 2(I) accumulation during liver brogenesis is similar regardless of the etiology of chronic liver injury. However, ethanol-induced liver brosis exhibits some special characteristics: acetaldehyde induces the upregulation of collagen 1(I) and 2(I) in HSCs [4951]. In particular, acetaldehyde activates transcription of both collagen 1(I) and 2(I) genes via the collagen transcription factor p35/EBP [52, 53]. In activated HSCs, the steady-state level of collagen 1(I) mRNA increases 60- to 70-fold compared to quiescent HSCs. While the transcription rate increases about 3-fold, the half-life of collagen 1(I) mRNA increases 16fold from 1.5 h in quiescent HSCs to 24 h in activated HSCs [54]. This indicates a major role for post-transcriptional regulation. Increased stability of collagen 1(I) mRNA is mediated by sequences in the 3 untranslated region (UTR) of the transcript, which interacts with a conserved stem-loop structure at the 5 end of the collagen 1(I) mRNA [55]. The RNA-binding protein CP binds to the 1(I) collagen 3-UTR, stabilizes this RNA and blocks RNA degradation in activated but not in quiescent HSCs [56]. Moreover, the interaction of the protein TRAM2 with the Ca2+ pump of the endoplasmatic reticulum, SERCA2b, is essential for triple helical collagen folding [57]. The 5-stem loop of the collagen 1(I) mRNA is further required to form triple helical collagen brils [57, 58]. After post-translational modications, type I collagen molecules are packed into brils in the ECM. The formation of covalent intermolecular cross-links of

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collagen is the nal modication step of collagen brils. This process provides the brils with mechanical strength. In cirrhotic livers, the mature non-reducible collagen cross-links are signicantly higher than in normal liver. Therefore, turnover and degradation of collagen is impaired in cirrhotic livers resulting in net collagen accumulation [48]. The proteolytic mechanisms of matrix degradation, which are a key feature of the ECM homeostasis in normal liver, are repressed and impaired during liver brogenesis. An altered matrix metalloproteinase activity leads to remodeling of hepatic ECM and accelerates brogenesis by increased net ECM production and HSC activation. HSCs are in particular a key source of matrix metalloproteinase-2 (MMP-2) and stromelysin/MMP-3, which degrade normal subendothelial ECM [5964], thereby promoting replacement by and accumulation of bril-forming collagen, which further increases HSC proliferation and MMP-2 production in a positive feedback loop [63, 65]. Through upregulation of inhibitory proteins, the tissue inhibitor of metalloproteinase-1 and -2 (TIMP-1 and -2), activated HSCs inhibit the activity of interstitial collagenases, thus additionally favoring accumulation of scar tissue [6062]. The most important stimulus for ECM production by HSCs in all chronic liver diseases including alcohol-induced liver brosis is TGF-. TGF- production is markedly increased in experimental and human hepatic brosis [11]. The cytokine is initially secreted by damaged hepatocytes, activated Kupffer cells or platelets. Especially its isoform TGF-1 is probably the most powerful and widely distributed pro-brogenic mediator in the body. Deposition of ECM is part of a TGF--dependent physiological wound-healing response to tissue injury that may progress to pathological brosis when the damaging agent is chronically provided [66]. TGF- induces expression of brillar and nonbrillar collagens, other interstitial matrix components including bronectin and tenascin, the basement membrane components laminin and entactin, and membrane proteoglycans including perlecan and biglycan [67]. Thus, TGF- contributes to ECM accumulation and irregular deposition in brotic tissue, e.g. by forming basal membranes that cause defenestration of hepatic sinusoids. Moreover, TGF- signaling decreases expression of various MMPs, which leads to hampered degradation and acceleration of ECM accumulation [68]. Transcriptional upregulation of the TGF- gene and of its cell membrane receptors is one feature of cultureactivated HSCs [69]. In addition, TGF- activity is enhanced in activated HSCs through proteolytic transfor-

mation of latent TGF- into the active cytokine by a urokinase-type plasminogen activator [70, 71]. Release and activity of TGF- are controlled by a number of intracellular and ECM-deposited interacting proteins. For example, plasmin treatment of cultured HSCs induces release of TGF- from the matrix accompanied by an increased TGF--dependent gene response, e.g., upregulation of Smad7 mRNA expression [72], indicating that plasmin participates in the activation process of latent TGF-. Furthermore, platelet-derived growth factor-BB-induced thrombospondin-1 expression enhances TGF- effects in HSCs [73]. TGF- signaling occurs after binding to its type I and II receptors [7476]. In HSCs it became evident that TGF- signaling is modulated during transdifferentiation from quiescent to activated HSCs. In quiescent HSCs, TGF- leads to phosphorylation of the transcription factor-like intracellular proteins Smad2 and Smad3 through a serine/threonine kinase-mediated process. Smad2 and 3 form oligomeric complexes with Smad4, which then translocate into the nucleus. This complex activates the TGF- reporter construct (CAGA)9-MLPluciferase, and leads to transcription of target genes, such as 1 and 2(I) collagen, or Smad7 [7779]. Smad7 acts as an inhibitor of this mechanism [80, 81]. Moreover, this TGF- pathway decreases the DNA synthesis in quiescent or early activated HSCs [77]. These acute effects of TGF- do not occur in fully activated HSCs, in which TGF-/TRII/Alk5 complex formation and TGF--dependent Smad2/3 activation and Smad7 induction are signicantly reduced. Interestingly, Smad3 is increased [78] and constitutively phosphorylated by p38 MAPK, a mechanism that contributes to ECM production in activated HSCs, both in vitro and in vivo [82]. A major role for Smad3 in brogenesis was also shown in carbon tetrachloride (CCl4)-treated Smad3-decient mice, providing that maximal expression of collagen type I and inhibition of proliferation, but not HSC activation requires Smad3 in HSCs, as assessed by the expression of the specic activation marker -smooth muscle actin (-SMA) [83]. Further studies indicate that Smad2 and Smad3 signal via independent pathways in HSCs and that Smad3 plays an important role in the morphological and functional maturation of hepatic myobroblasts [84]. In alcoholic liver brosis, acetaldehyde elevates steadystate levels of TGF- mRNA [50], promotes activation of latent TGF- protein and upregulates the expression of its receptor type II (TRII) [85]. Reducing TGF- protein levels causes a decrease in acetaldehyde-induced collagen 2(I) gene transcription in activated HSC [42, 86],

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whereas early acetaldehyde-dependent upregulation of collagen is independent of TGF- and modulated by PI3kinase [51].

Novel Molecular Anti-Fibrotic Treatment Strategies

While avoiding the damaging agent, e.g. alcohol, still remains the most important healing approach, efforts have been undertaken to blunt the process of brogenesis at the molecular level or to specically induce cell death in activated HSCs. Many of the novel molecular therapeutic approaches target the most prominent probrogenic signaling pathway, initiated by TGF-. For example, adenoviral overexpression of dominant negative TRII attenuated dimethylnitrosamine (DMN)-induced liver brosis in rats [87]. Moreover, an adenovirus expressing an entire ectodomain of human TRII fused to the Fc portion of human IgG was similarly efcient in the DMN model without apparent systemic or local side effects [88]. This result indicates that the transient systemic presence of soluble TRII is sufcient to inhibit TGF1 action and might therefore be a useful approach for the treatment of different diseases that involve TGF- signaling without the necessity to directly target the affected organ. Another strategy of TGF- neutralization is targeting the activation process of latent TGF-. Thus, the serine protease inhibitor camostat mesilate attenuated hepatic brogenesis by inhibiting plasmin activity [71]. Further, blockade of thrombospondin-1, an ECM protein that activates the small latent TGF- complex, prevented progression of hepatic damage and brosis in a DMN model in vivo [89]. We used a more specic downstream blockade of the TGF- signaling pathway by overexpression of Smad7 [80]. In a rat model of bile duct ligation-induced liver brosis, injections of an adenovirus carrying Smad7 cDNA into the portal vein during surgery and via the tail vein at later stages lead to reduced hepatic collagen expression and hydroxyproline content. In addition, -SMA staining was strongly reduced in animals treated with the Smad7 expression cassette compared to controls, indicating a signicant reduction of activated HSC in vivo. Importantly, such a benecial effect was also observed when Smad7 was expressed in animals with established brosis. Further in vitro experiments in HSCs indicated that the underlying mechanisms most likely involve inhibition of TGF- signaling and HSC transdifferentiation. Additionally, Smad7 may also have other, yet unidentied functions, that may

also contribute to the observed effects. Therefore, further research on the details of TGF- signaling and the impact of Smad7 in the different cell types of the liver is needed to optimize treatment strategies based on Smad7. According to various experimental models, IFN- has the potential to antagonize several TGF- effects. We recently investigated the impact of IFN- on hepatic brosis in patients with chronic hepatitis B viral infection with emphasis on IFN- signaling during activation of HSCs [90]. Histology and serum indices displayed lower brosis scores and liver damage in the IFN- treatment group. In parallel, the number of -SMA-positive cells was strongly downregulated in patients treated with IFN-. Since it is known from other trials that IFN-, in contrast to other interferons, does not display signicant antiviral effects, we investigated the impact of this cytokine on liver brogenesis in HSCs. Transfection of a Smad7 promoter construct and Smad7 immunoblotting showed that IFN- was able to induce Smad7 transcription and protein expression. Furthermore, our data revealed that IFN- signaling in HSCs is mediated via STAT-1 activation. Nuclear phospho-Smad2 staining was predominant in damaged tissue and absent after IFN- treatment, whereas strong cytoplasmatic Smad7 staining was only observed after IFN- application. In conclusion, our data indicate that disease-dependent TGF- signaling is abrogated by IFN- treatment. The resolution of liver brosis correlates with an increase in HSC death [4, 91]. HSC apoptosis is associated with reduced TIMP-1 expression and has been documented during the recovery phase of experimentally induced liver injury [91]. Stellate cells also undergo spontaneous apoptosis during activation, in parallel with increased expression of death receptors of the TNF receptor superfamily and their ligands, or proapoptotic proteins, such as p53, mediating their programmed cell death [9294]. Current treatment strategies for liver brosis also include the selective induction of cell death in HSCs [95]. In particular, members of the TNF-receptor superfamily including TNF-, TNF-related apoptosis-inducing ligand, nerve growth factor and Fas ligand have been shown to induce cell death in HSCs [94, 9698], but are not HSC-specic. In contrast, gliotoxin, a toxin produced by Aspergillus fumigatus, causes apoptosis in HSCs at low concentrations, but not in hepatocytes [99101] and has been shown to reduce liver brosis in vivo [99, 102]. Gliotoxin induces formation of ROS in HSCs, and mediates cell death via inhibition of NFB, mitochondrial permeability transition, cytochrome c release and caspase-3 activation [100, 101]. Hepatocytes are unaffected by glio-

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toxin at low concentrations due to their ability to quickly and efciently metabolize the substance [101]. Recently, we have shown that anandamide, an endocannabinoid, also selectively inhibits HSC proliferation at lower concentrations and induces HSC death at higher concentrations [39]. Anandamide mediates cell death in HSCs through the interaction with membrane cholesterol, entailing an increase in intracellular ROS and inux of calcium. Hepatocytes are resistant to anandamide-induced cell death due to lower binding levels compared to HSCs and due to expression of the anandamide-degrading enzyme, fatty acid amide hydrolase, which is present in HSCs only in trace amounts. We also examined the effect of TIMP-1 inactivation in hepatic brosis through administration of a human anti-rat TIMP-1 antibody in a rat model of CCl4-induced brosis [103]. Rats treated with the TIMP-1 antibody had reduced levels of liver brosis compared to controls, as determined by histological analysis and hydroxyproline content. In addition, the antibody caused reductions in MMP-2 activity and -SMA expression. We also observed a reduction in the number of activated HSCs. This was presumably due to induction of apoptosis in HSCs, since TIMP-1 exerts anti-apoptotic effects in vitro and in vivo [104, 105]. Our results of this new therapeutic approach are particularly encouraging, because brosis was established before the administration of the antibody, and rats continued to receive CCl4 simultaneously with therapy, thus better reecting the clinical approach of liver brosis treatment. Other potential candidates for molecular therapeutic approaches for ALD are summarized in Breitkopf et al. [106] and Siegmund and Brenner [107].

From Alcoholic Liver Fibrosis to Cirrhosis Clinical Consequences

Liver cirrhosis is the end stage of ALD and is dened as an irreversible remodeling of the normal liver architecture with diffuse and bridging brosis, loss of vascular cross-sectional area and irregular nodular regeneration of hepatocellular parenchyma. The irregular hepatic architecture occurs due to: (i) the continuing hepatocellular cell death due to persistent alcohol consumption and ongoing inammation; (ii) the excessive accumulation of ECM scar mass by activated HSCs; (iii) the increased hepatocellular regeneration due to the hepatocytes distinctive ability to proliferate, and (iv) the rearrangement of the hepatic microvasculature [2].

Interestingly, alcoholic liver cirrhosis features mostly micronodular regeneration of hepatocellular tissue [108], also known as Lannecs cirrhosis (g. 1). One explanation of this morphological aspect may be the fact that ethanol actively inhibits liver regeneration by blocking the signaling cascades of growth factors, such as epidermal growth factor and insulin or by upregulating the expression of anti-proliferative and pro-brogenic cytokines such as TGF- [109]. Ethanol withdrawal after manifestation of liver cirrhosis consequently leads to a more macronodular morphology of the cirrhotic liver [108]. Interestingly, in spite of ethanols effect in inhibition of hepatocyte regeneration, chronic ethanol consumption causes an increased expression of the proto-oncogene protein c-myc as well as DNA hypomethylation. Both conditions account for the higher risk of the development of hepatocellular carcinoma in alcoholic liver cirrhosis [25]. The disruption of the normal anatomy of the liver lobule by the accumulation of hepatic scar tissue in combination with the defenestration of the sinusoidal epithelial lining due to the development of manifest basal membranes in the subsinusoidal space of Diss leads to impaired transsinusoidal exchange of substances. The result is further hepatocellular hypoxia followed by aggravated loss of hepatic parenchyma [2]. This pathophysiological mechanism applies equally to substances that are transported into the liver by the portal blood and that are released by hepatocytes into the sinusoids. Thus, the decreasing hepatic ability to detoxify noxious substances leads to their accumulation in the circulation (e.g. ammonia). The impaired ability for detoxication may even contribute to increased ethanol toxicity during this stage of ALD, if ethanol abuse is still present. It also accounts for the decreased ability of the liver to generate and secrete indispensable metabolites (e.g. coagulation factors). Clinical consequences include hepatic encephalopathy and elevated hemorrhagic diathesis. Furthermore, the massive alterations in hepatic microcirculation caused by hepatic brosis are a major reason for further clinical complications such as portal hypertension, formation of esophageal, gastric or rectal varices with the risk of lifethreatening hemorrhage, portal hypertensive gastropathy, ascites, and hepatorenal syndrome [110]. The pathophysiological mechanism of these complications comprises the signicant diminution of overall vascular cross-sectional area. Perisinusoidal brosis causes narrowing or occlusion and concomitant loss of sinusoids [111, 112]. On the other hand, porto-caval shunt vessels occur during progressive brosis that conduct the portal

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blood around the newly formed parenchymal nodules without any noticeable exchange of macromolecular metabolites [2]. Additionally, these vessels contribute to increased portal pressure [2]. Another reason for decreasing vascular capacity are activated HSCs themselves, which develop a remarkable contractile ability [10, 11, 14]. HSC contractility represents an important mechanism for the development of increasing portal resistance during liver injury. The key contractile stimulus toward stellate cells is mainly autocrine-derived endothelin (ET)-1, which also represents a highly proliferative cytokine for activated HSCs [113, 114]. Upregulation of ET-1 production is accompanied by increased endothelin-converting enzyme-1, which activates the latent ET-1 [115]. On the other hand, autocrine and paracrine mediators that are reducing portal blood pressure, such as nitric oxide and prostaglandin E2, are downregulated during brogenesis [95, 116]. Moreover, during the development of liver cirrhosis, angiogenesis is increased with deleterious effects on hepatic hemodynamics. VEGF receptors are upregulated after injury in both sinusoidal endothelial cells and stellate cells [40, 41]. Stimulation of these receptor tyrosine kinases induces angiogenesis. In hypoxic conditions, such as ALD, both VEGF and its receptor mRNAs are rapidly induced in stellate cells, establishing an autocrine and paracrine loop supporting the development of new blood vessels [41]. It becomes evident that the molecular aspects of brogenesis, the loss of hepatocellular function and the vascular modications during the development of cirrhosis account for the entire development of the pathological condition. Unfortunately, the therapeutic opportunities in this nal stage are still very limited, and have to be rather symptomatic than causative [95]. In most cases, liver transplantation remains the only option left to save a patients life [117].

Conclusions

Alcohol abuse accounts for more than half of the prevalence of liver brosis and cirrhosis in the Western world. Fibrosis of the liver is a wound-healing process that occurs due to persistent hepatocellular injury. Although the major mechanisms of brogenesis are independent of the origin of liver injury, alcoholic liver brosis features several special characteristics. An important aspect in ALD is the pronounced inammatory response of Kupffer cells and other types of leukocytes (macrophages, neutrophils, lymphocytes), due to elevated gut-derived endotoxin

plasma levels. This leads to amplied formation of ROS and cell-toxic or pro-brogenic cytokines (e.g. TNF- or TGF-1, respectively), which are together responsible for increased hepatocellular cell death and activation of HSCs, the key cell type of liver brogenesis. HSCs change from quiescent, vitamin A-storing cells into myobroblast-like, collagen-producing cells. HSC activation is a complicated process that involves a network of diverse mediators and cell types which activate HSCs. Moreover, ethanol metabolism induces hypoxia in the pericentral region of the liver lobule causing rst hepatocellular damage and accumulation of scar tissue at this site. Ethanol metabolites, such as acetaldehyde, aldehyde-protein adducts or lipid oxidation products directly enhance HSC activation and production of brillar collagen. If the injurious driving force, e.g. ethanol, is not withdrawn in time, the liver becomes cirrhotic. Liver cirrhosis features a remodeling of the normal liver architecture including brosis, loss of vascular space and irregular nodular regeneration of hepatocytes. Alcoholic liver cirrhosis is mostly micronodular because of the inhibitory action of ethanol on hepatocyte growth. Destruction of the regular liver architecture accounts for the clinical consequences of liver cirrhosis. Loss of functional hepatocellular parenchyma leads to decreasing hepatic detoxication of, e.g., ammonia, acetaldehyde or ethanol, and to impaired hepatic metabolization of, e.g., coagulation factors or albumin. Liver brosis with built-up of basal membranes, brotic septa and loss of sinusoidal fenestration signicantly contributes to this pathophysiological mechanism. Portal hypertension as another crucial cause of clinical complications, such as the occurrence of esophageal varices with the danger of life-threatening hemorrhage, is a consequence of the rearrangement of the hepatic vascular bed. Although withdrawal of ethanol still is the most effective intervention to prevent the manifestation of alcoholic liver cirrhosis, molecular approaches are underway that target probrogenic signal transduction in the liver or aim to induce cell death specically in activated HSCs. For patients with cirrhosis and clinical complications, liver transplantation is currently the only curative approach.

Acknowledgements
S.V.S. is supported by a grant from the Research Fund of the Faculty of Clinical Medicine Mannheim, University of Heidelberg, Germany (Formatstipendium). D.A.B. is supported by grants from the NIH. S.D. is supported by the Dietmar Hopp Foundation, Walldorf, Germany.

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